Adapting the polymerase chain reaction to a double-stranded RNA genome.
We have adapted the polymerase chain reaction (PCR) to a double-stranded RNA (dsRNA) target without possessing unambiguous sequence information. Infectious bursal disease virus of chickens, a member of the binavirus group, has a dsRNA genome which is resistant to denaturation and subsequent enzyme modification. The only published sequence information was for a strain of virus unavailable to us. We have used a quick primer binding assay to select appropriate primers and have combined a simple denaturation method with reverse transcription and subsequent polymerization using the cDNA template to yield amplified product easily detectable by ethidium bromide staining. By varying the times of denaturation, annealing, and polymerization and by reducing the total number of amplification cycles, artifacts have been eliminated when using purified genome as the template. This allowed us to obtain partial sequence information for one viral strain. We have enhanced the utility of our method by optimizing a rapid cell lysis and capsid digestion protocol such that no purification steps are required from initial tissue handling through final PCR product. Total time for all procedures involved no more than 6 h. This technique should be applicable to all other members of the Birnaviradae family and to any other species of dsRNA.
Davis
V S
VS
College of Veterinary Medicine, Mississippi State University, Mississippi State 39762.
Boyle
J A
JA
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002645:Chickens; D015698:Genomic Library; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012330:RNA, Double-Stranded / Q000235:genetics*; D013698:Templates, Genetic
D009841:Oligonucleotides; D012330:RNA, Double-Stranded